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M Rola-Pleszczynski

Publications and source records attributed to M Rola-Pleszczynski.

At least 19 recordsLinked to original sources
PubMed2001

IL-13 and IL-4 up-regulate cysteinyl leukotriene 1 receptor expression in human monocytes and macrophages.

The cysteinyl (Cys) leukotrienes (LT)C(4), LTD(4), and LTE(4), are lipid mediators that have been implicated in the pathogenesis of asthma. The human LTD(4) receptor (CysLT(1)R) was recently cloned and characterized. The present work was undertaken to study the potential modulation of CysLT(1)R expression by the Th2 cytokines IL-13 and IL-4. In this study, we report that IL-13 up-regulates CysLT(1)R mRNA levels, with consequently enhanced CysLT(1)R protein expression and function in human monocytes and monocyte-derived macrophages. CysLT(1)R mRNA expression was augmented 2- to 5-fold following treatment with IL-13 and was due to enhanced transcriptional activity. The effect was observed after 4 h, was maximal by 8 h, and maintained at 24 h. IL-4, but not IFN-gamma, induced a similar pattern of CysLT(1)R up-regulation. Monocytes pretreated with IL-13 or IL-4 for 24 h showed enhanced CysLT(1)R protein expression, as assessed by flow cytometry using a polyclonal anti-CysLT(1)R Ab. They also showed enhanced responsiveness to LTD(4), but not to LTB(4), in terms of Ca(2+) mobilization, as well as augmented chemotactic activity. Our findings suggest a possible mechanism by which IL-13 and IL-4 can modulate CysLT(1)R expression on monocytes and macrophages, and consequently their responsiveness to LTD(4), and thus contribute to the pathogenesis of asthma and allergic diseases.

Asthma↗
PubMed2001

G-protein-independent activation of Tyk2 by the platelet-activating factor receptor.

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid with multiple physiological and pathological effects. PAF exerts its activity through a specific heptohelical G-protein coupled receptor, expressed on a variety of cell types, including leukocytes. In this study, we showed that PAF induced a rapid tyrosine phosphorylation of the Tyk2 kinase in the monocytic cell lines U937 and MonoMac-1. PAF-initiated Tyk2 phosphorylation was also observed in COS-7 cells transiently transfected with the human PAF receptor (PAFR) and Tyk2 cDNAs. In addition, we found that Tyk2 co-immunoprecipitated and co-localized with PAFR, independently of ligand binding. Deletion mutants of Tyk2 indicated that the N terminus of the kinase was important for the binding to PAFR. Activation of Tyk2 was followed by a time-dependent 2-4-fold increase in the level of tyrosine phosphorylation of signal transducers and activators of transcription 1 (STAT1), STAT2, and STAT3 and a sustained 2.5-fold increase in STAT5 tyrosine phosphorylation. In MonoMac-1 cells, STAT1 and STAT3 translocated to the nucleus following PAF stimulation, and their translocation in transiently transfected COS-7 cells was shown to be dependent on the presence of Tyk2. In addition, when COS-7 cells were transfected with PAFR and constructs containing PAFR promoter 1, coupled to the luciferase reporter gene, PAF induced a 3.6-fold increase in promoter activation in the presence of Tyk2. Finally, PAFR mutants that could not couple to G-proteins were found to effectively mediate Tyk2 activation and signaling. Taken together, these findings suggest an important role for the Janus kinase/STAT pathway in PAFR signaling, independent of G-proteins, and in the regulation of PAF receptor expression by its ligand.

Active Transport, Cell Nucleus↗
PubMed2001

Inverse agonist activity of selected ligands of platelet-activating factor receptor.

The receptor for platelet-activating factor (PAFR) is a member of the G protein-coupled receptor (GPCR) family. According to the allosteric ternary complex model, GPCRs exist in an equilibrium between different conformations. Agonist binding promotes and stabilizes the receptor in an active conformation. On the other hand, ligands that stabilize the inactive conformation are known as inverse agonists. Due to the association of platelet-activating factor (PAF) with diverse physiological and pathological processes, considerable efforts have been invested in the development of antagonists to PAFR. A large number of these molecules has been shown to specifically interact with PAFR but, surprisingly, little is known about their impact on the conformation of the receptor and its activity. By using a constitutively active mutant (L231R) of the human PAFR and by transiently coexpressing the wild-type (WT) receptor with the G(alpha)q subunit of the trimeric G protein, we were able to address this issue with ligands of diverse structures such as phospholipids, benzodiazepines, furans, and others. We demonstrated that some of these molecules are potent inverse agonists. For example, when cells (WT PAFR + G(alpha)q) were exposed to WEB2086, SM10661, or alprazolam, the basal inositol phosphate production was reduced by 53 +/- 6, 44 +/- 3, and 54 +/- 4%, respectively. The decrease in basal inositol phosphate production by WEB2086 was significantly inhibited by a more neutral antagonist BN52021, confirming the specificity of the reaction. We demonstrate here that WEB2086 and other known ligands previously considered as antagonists can act as inverse agonists on the human PAF receptor.

Animals↗
PubMed2000

Expression of platelet-activating factor receptor in human carotid atherosclerotic plaques: relevance to progression of atherosclerosis.

BACKGROUND: Human monocyte-derived macrophages synthesize numerous proinflammatory and prothrombotic substances, including lipid mediators, such as platelet-activating factor (PAF), which may play a major role in the onset and perpetuation of atherosclerotic lesions. In addition, both monocytes and macrophages express PAF receptors (PAF-R). The expression of PAF-R is transcriptionally downregulated by oxidized LDL in in vitro primary cultures of monocyte/macrophages. In this study, we evaluated the expression of PAF-R in human carotid plaque tissue, in foam cells isolated from human carotid plaques, and in primary cultures of umbilical smooth muscle cells (SMCs). METHODS AND RESULTS: We show that PAF-R was expressed at low levels in foam cells compared with monocyte/macrophages in plaques, as assessed by immunohistochemical staining and in situ hybridization. In addition, low levels of mRNA were also detected by RT-PCR in isolated human carotid foam cells. A prominent finding of our study was the demonstration that contractile SMCs were positive for PAF-R, and its mRNA was extracted from primary cultures of umbilical SMCs. CONCLUSIONS: As macrophages loose their inflammatory phenotype on transformation into foam cells, they may equally loose their capacity of defense against aggression. We postulate that the diminished expression of PAF-R may be deleterious in the context of plaque formation and progression. The observation that arterial SMCs of contractile phenotype express PAF-R opens new avenues concerning the migration of these cells from media to intima and atherosclerotic plaque formation.

Antigens, Differentiation↗
PubMed2000

IL-5 up-regulates cysteinyl leukotriene 1 receptor expression in HL-60 cells differentiated into eosinophils.

The cysteinyl leukotrienes, leukotriene (LT) C(4), LTD(4), and LTE(4), are lipid mediators that have been implicated in the pathogenesis of several inflammatory processes, including asthma. The human LTD(4) receptor, CysLT(1)R, was recently cloned and characterized. We had previously shown that HL-60 cells differentiated toward the eosinophilic lineage (HL-60/eos) developed specific functional LTD(4) receptors. The present work was undertaken to study the potential modulation of CysLT(1)R expression in HL-60/eos by IL-5, an important regulator of eosinophil function. Here, we report that IL-5 rapidly up-regulates CysLT(1)R mRNA expression, with consequently enhanced CysLT(1)R protein expression and function in HL-60/eos. CysLT(1)R mRNA expression was augmented 2- to 15-fold following treatment with IL-5 (1-20 ng/ml). The effect was seen after 2 h, was maximal by 4 h, and maintained at 8 h. Although CysLT(1)R mRNA was constitutively expressed in undifferentiated HL-60 cells, its expression was not modulated by IL-5 in the absence of differentiation. Differentiated HL-60/eos cells pretreated with IL-5 (10 ng/ml) for 24 h showed enhanced CysLT(1)R expression on the cell surface, as assessed by flow cytometry using a polyclonal anti-CysLT(1)R Ab. They also showed enhanced responsiveness to LTD(4), but not to LTB(4) or platelet-activating factor, in terms of Ca(2+) mobilization, and augmented the chemotactic response to LTD(4). Our findings suggest a possible mechanism by which IL-5 can modulate eosinophil functions and particularly their responsiveness to LTD(4), and thus contribute to the pathogenesis of asthma and allergic diseases.

Blotting, Northern↗
PubMed2000

PAF produced by human breast cancer cells promotes migration and proliferation of tumor cells and neo-angiogenesis.

Platelet-activating factor (PAF), a phospholipid mediator of inflammation, is present in breast cancer tissue and correlates with microvessel density. In the present study, we investigated the biological significance of PAF synthesized within breast cancer. In vitro, we observed the production of PAF by two estrogen-dependent (MCF7 and T-47D) and an estrogen-independent (MDA-MB231) breast cancer cell lines after stimulation with vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, tumor necrosis factor, thrombin but not with estrogen, progesterone, and oxytocin. The sensitivity to agonist stimulation and the amount of PAF synthesized as cell-associated or released varied in different cell lines, being higher in MDA-MB231 cells, which are known to be highly invasive. We further demonstrate, by reverse transcriptase-polymerase chain reaction and cytofluorimetry, that all of the breast cancer cells express the PAF receptor and respond to PAF stimulation in terms of proliferation. Moreover, in MDA-MB231 cells PAF elicited cell motility. In vivo, two structurally different PAF receptor antagonists WEB 2170 and CV 3988 significantly reduced the formation of new vessels in a tumor induced by subcutaneous implantation of MDA-MB231 cells into SCID mice. In conclusion, these results suggest that PAF, produced and released by breast cancer cells, can contribute to tumor development by enhancing cell motility and proliferation and by stimulating the angiogenic response.

Animals↗
PubMed1999

Selective modulation of wild type receptor functions by mutants of G-protein-coupled receptors.

Members of the G-protein-coupled receptor (GPCR) family are involved in most aspects of higher eukaryote biology, and mutations in their coding sequence have been linked to several diseases. In the present study, we report that mutant GPCR can affect the functional properties of the co-expressed wild type (WT) receptor. Mutants of the human platelet-activating factor receptor that fail to show any detectable ligand binding (N285I and K298stop) or coupling to a G-protein (D63N, D289A, and Y293A) were co-expressed with the WT receptor in Chinese hamster ovary and COS-7 cells. In this context, N285I and K298stop mutant receptors inhibited 3H-WEB2086 binding and surface expression. Co-transfection with D63N resulted in a constitutively active receptor phenotype. Platelet-activating factor-induced inositol phosphate production in cells transfected with a 1:1 ratio of WT:D63N was higher than with the WT cDNA alone but was abolished with a 1:3 ratio. We confirmed that these findings could be extended to other GPCRs by showing that co-expression of the WT C-C chemokine receptor 2b with a carboxyl-terminal deletion mutant (K311stop), resulted in a decreased affinity and responsiveness to MCP-1. A better understanding of this phenomenon could lead to important tools for the prevention or treatment of certain diseases.

Animals↗
PubMed1999

Modulation of formyl peptide receptor expression by IL-10 in human monocytes and neutrophils.

IL-10, originally described as a cytokine synthesis inhibitory factor, is secreted by a number of cells of the immune system, including monocytes and T cells. Although IL-10 is being assigned as an immunosuppressive cytokine, our study showed that FMLP-R mRNA was rapidly up-regulated by exposure of monocytes to graded concentrations of this cytokine, with maximal (three- to fourfold) stimulation with 10 ng/ml. The effect was rapid, being observable as early as 1 h of treatment with IL-10, maximal between 2 and 4 h, and still evident after 24 h and was associated with an increase of receptor expression on the cell surface as assessed by flow cytometry analysis. Pretreatment of monocytes with actinomycin D completely abrogated the effect of IL-10, suggesting a transcriptional regulation. Moreover, IL-10-treated monocytes showed a significantly enhanced functional responsiveness to FMLP with enhanced (three- to fourfold) chemotaxis and augmented (twofold) intracellular calcium mobilization. In polymorphonuclear neutrophils (PMN), IL-10 also mediated a twofold augmentation of FMLP-R expression. In parallel experiments, we observed that IL-10 could differentially modulate other chemotactic receptors. Hence, we observed that IL-10 augmented two-to threefold platelet-activating factor receptor (PAF-R) expression, whereas it had no significant effect on the fifth component of complement (C5a) receptor (C5a-R) expression. Collectively, our results demonstrate that IL-10 may play an important role in inflammatory process through modulation of chemotactic receptor expression.

Chemotaxis, Leukocyte↗
PubMed1999

IL-10 up-regulates CCR5 gene expression in human monocytes.

The chemokine receptor CCR5 has been found to play a key role in early infection with macrophage-tropic isolates of HIV-1 and CCR5 deficiency is associated with a relative resistance to HIV-1 infection. In this context, we studied the regulation of CCR5 gene expression by cytokines, and in particular, interleukin (IL)-10 in human monocytes. CCR5 mRNA was rapidly up-regulated by exposure of monocytes to graded concentrations of IL-10, with near maximal stimulation with 10 ng/ml. The effect was rapid, being detectable as early as 1 h and maximal between 2 and 4 h of treatment. Pretreatment of monocytes with actinomycin D prevented the IL-10-induced effect, suggesting a transcriptional mechanism for CCR5 up-regulation by IL-10. Protein expression of CCR5 was also enhanced by IL-10, as indicated by a 3-fold increase in anti-CCR5 antibody labeling of monocytes treated for 20 h with IL-10. Increased surface expression of CCR5 persisted at 48 h of treatment. Moreover, IL-10-treated monocytes responded with augmented intracellular Ca++ mobilization and enhanced (3-4-fold) chemotaxis in response to the CCR5 ligand MIP-1beta(25 ng/ml). Taken together, our data indicate that IL-10 can modulate CCR5 expression and function. This can constitute a potentially important regulatory mechanism which can affect not only responses during inflammation, but also susceptibility to HIV-1 infection.

Antibodies, Monoclonal↗
PubMed1998

Signalling through the leukotriene B4 receptor involves both alphai and alpha16, but not alphaq or alpha11 G-protein subunits.

COS-7 cells transfected with the leukotriene (LT) B4 receptor (BLTR) cDNA were unable to produce LTB4-induced inositol phosphates (IPs) in spite of the presence of endogenous Galphai, Galphaq and Galpha11 proteins. Co-transfection of BLTR with Galpha16, however, resulted in high levels of IP production, which were 17-, 10- and 6-fold higher than with co-transfected Galpha11, Galphaq and Galpha14, respectively. Co-transfection of BLTR with phospholipase C (PLC) beta2, on the other hand, resulted in efficient IP production and co-transfection of BLTR with both Galpha16 and PLCbeta2 resulted in a greater than additive response.

Animals↗
PubMed1998

Prostaglandin E2 induces resistance to human immunodeficiency virus-1 infection in monocyte-derived macrophages: downregulation of CCR5 expression by cyclic adenosine monophosphate.

The chemokine receptor CCR5 can function as a coreceptor for human immunodeficiency virus-1 (HIV-1) entry into CD4(+) T cells and macrophages, especially during the early stages of HIV-1 infection. The regulation of CCR5 expression may affect not only leukocyte migration, but also infectivity by HIV-1 and, therefore, acquired immunodeficiency syndrome (AIDS) pathogenesis. We report here that agents which increase intracellular concentrations of cyclic adenosine monophosphate (cAMP) rapidly downregulate CCR5 gene expression, with consequent loss of CCR5 expression and function in monocytes/macrophages. Chemotaxis and intracellular Ca2+ mobilization in monocytes pretreated with prostaglandin E2 or dibutyryl-cAMP for 24 hours were significantly reduced in response to the CCR5 ligand, MIP-1beta. Moreover, HIV-1 entry into monocyte-derived macrophages pretreated with dibutyryl-cAMP or prostaglandin E2 was markedly decreased. Our findings suggest that resistance to HIV-1 can be induced by agents which increase cellular levels of cAMP and that this may suggest additional therapeutic strategies to limit infection by HIV-1.

Cyclic AMP↗
PubMed1998

Localization, quantification, and activation of platelet-activating factor receptor in human endometrium during the menstrual cycle: PAF stimulates NO, VEGF, and FAKpp125.

Implantation is characterized by an inflammatory-like response with expansion of extracellular fluid volume, increased vascular permeability, and vasodilatation. These effects are believed to be mediated at the paracrine level by prostaglandin E2 and platelet-activating factor (PAF), but the cellular mechanism (or mechanisms) remains largely unknown. We demonstrate that PAF receptor (PAF-R) immunoreactivity and mRNA are detected in proliferative and secretory endometrial glands, however, the responsiveness of endometrium to physiological concentrations of PAF is confined predominantly to the secretory endometrium. Semiquantitative reverse transcription-polymerase chain reaction revealed that PAF-R transcript levels were highest in the mid-late proliferative and late secretory phases of the cycle. Interaction of PAF with its receptor resulted in the rapid release of nitric oxide (NO), increased expression of vascular endothelial growth factor (VEGF), and activation of FAKpp125, a focal adhesion kinase, demonstrating that the PAF-R is functionally active. Inhibition of NO synthesis by NG-monomethyl-L-arginine produced dose-dependent attenuation of PAF-evoked NO release, indicating NOS activation; the dependency of PAF-evoked NO release on PKC and extracellular Ca2+ was confirmed by PKC inhibitor Ro 31-8220 and by the removal of extracellular Ca2+. PAF up-regulated VEGF gene expression in a concentration- and time-dependent fashion in human endometrial epithelial cell lysates. Transcription of VEGF was rapidly followed by secretion of the protein. These data support our premise that this autocoid acts as an angiogenic mediator in the regeneration of the endometrium after menses and as a vasodilator to promote blastocyst attachment during the implantation process.

Cell Adhesion Molecules↗
PubMed1997

Epstein-Barr virus induces GM-CSF synthesis by monocytes: effect on EBV-induced IL-1 and IL-1 receptor antagonist production in neutrophils.

Neutrophils play an important role in the control of viral infections by releasing a variety of potent agents. We previously demonstrated that Epstein-Barr virus (EBV) binds to human neutrophils and stimulates cytokine synthesis including interleukin-1 (IL-1) and IL-1 receptor antagonist (IL-1Ra). Since neutrophil functions are known to be modulated by the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), we therefore investigated the cellular source of GM-CSF synthesis following treatment of leukocytes with EBV and the effect of GM-CSF on the production of IL-1, IL-1Ra, and superoxide by EBV-treated neutrophils. In enriched-cell populations, only monocytes were found to produce GM-CSF in response to EBV, which was maximal after 12 h of incubation. The results obtained with UV-irradiated particles or EBV neutralized with monoclonal antibody 72A1 suggest that contact between the cell and the gp350 of the viral envelope is sufficient to induce the release of GM-CSF. On the other hand, GM-CSF differentially upregulated EBV-induced IL-1 and IL-1Ra production by neutrophils. Pretreatment of neutrophils with GM-CSF prior to EBV activation synergistically enhanced the production of IL-1 alpha and IL-1 beta, but only marginally affected IL-1Ra synthesis. In addition, GM-CSF was also found to synergistically enhance the superoxide production by neutrophils in response to EBV. Molecular analysis showed that GM-CSF did not alter the IL-1 beta and IL-1Ra mRNA synthesis induced by EBV, suggesting that GM-CSF could act at a posttranslational level. Local production of GM-CSF by monocytes in tissues invaded by EBV could serve to potentiate the host defense mechanisms directed toward the destruction of the infectious virus.

Animals↗
PubMed1997

Structural and functional requirements for agonist-induced internalization of the human platelet-activating factor receptor.

The receptor for platelet-activating factor (PAF) is a member of the G-protein-coupled receptor family. To study the structural elements and mechanisms involved in the internalization of human PAF receptor (hPAFR), we used the following mutants: a truncated mutant in the C-terminal tail of the receptor (Cys317 --> Stop) and mutations in the (D/N)P(X)2,3Y motif (Asp289 --> Asn,Ala and Tyr293 --> Phe,Ala). Chinese hamster ovary cells expressing the Cys317 --> Stop mutant exhibited a marked reduction in their capacity to internalize PAF, suggesting the existence of determinants important for endocytosis in the last 26 amino acids of the cytoplasmic tail. Substitution of Asp289 to alanine abolished both internalization and G-protein coupling, whereas substitution of Tyr293 to alanine abolished coupling but not internalization. Inhibition or activation of protein kinase C did not significantly affect the internalization process. Receptor sequestration and ligand uptake was, at least in part, blocked by concanavalin A and blockers of endocytosis mediated by clathrin-coated pits. Our data suggest that the internalization of a G-protein-coupled receptor and coupling to a G-protein can be two independent events. Moreover, the C terminus tail of hPAFR, but not the putative internalization motifs, may be involved in the internalization of hPAFR.

Animals↗
PubMed1997

Role of the Cys90, Cys95 and Cys173 residues in the structure and function of the human platelet-activating factor receptor.

Platelet-activating factor (PAF) is a potent phospholipid mediator which binds to a specific, high affinity receptor of the G protein-coupled receptor family. In the present report, we show that ligand binding to the PAF receptor is sensitive to the reducing agent dithiothreitol (DTT), suggesting the involvement of disulfide linkages in the proper PAF receptor conformation. Substitutions of Cys90, Cys95 and Cys173 to Ala or Ser demonstrated that these cysteine residues are critical for normal cell surface expression of the PAF receptor protein and ligand binding to the receptor. The Cys90 and Cys173 mutant receptors did not display any specific ligand binding, were not expressed on the cell surface but were found in the intracellular compartment. The Cys95 mutants showed specific binding and were able to stimulate low levels of inositol phosphate (IP) production. These mutants were expressed at low density on the cell surface and showed high expression intracellularly. Our results suggest that the structure and function of the PAF receptor require the conserved Cys90 and Cys173 to form a disulfide bond. Moreover, Cys95 also appears to be necessary, possibly by establishing a disulfide linkage with an as yet unidentified Cys residue. All three residues appear essential for the proper folding and surface expression of the PAF receptor protein.

Amino Acid Sequence↗
PubMed1997

Augmented expression of platelet-activating factor receptor gene by TNF-alpha through transcriptional activation in human monocytes.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine produced by activated monocytes and often associated with platelet-activating factor (PAF) during the pathogenesis of many inflammatory and infectious diseases. PAFR is a G-protein-coupled receptor constitutively expressed on monocytes. TNF-alpha (100-400 U/mL) significantly increased PAFR mRNA expression in human monocytes. This increase was seen after 1 h of stimulation and persisted up to 24 h. Actinomycin D pretreatment studies revealed a transcriptional increase in PAFR gene expression without effect on mRNA half-life. [3H]WEB 2086 binding studies showed a significant (43%) increase in specific binding sites in 24-h-treated cells without change in receptor affinity. Increased interleukin-6 production in response to PAF was also found in 24-h TNF-alpha-pretreated monocytes. These observations provide new evidence for TNF-alpha and PAF interactions in human monocytes during inflammatory processes through up-regulation of PAFR expression by TNF-alpha.

Azepines↗
PubMed1997

Changes in apoptosis of human polymorphonuclear granulocytes with aging.

Many alterations with aging occur at the cellular and organic levels in the immune system ultimately leading to a decrease in the immune response. Our aim in the present work was to study apoptosis of polymorphonuclear granulocytes (PMN) with aging under various stimulations since apoptosis might play an important role in several pathologies encountered with aging. The PMN of healthy young (20-25 years) and elderly (65-85 years) subjects were examined after 24 h of sterile culture with and without stimulation. The stimulating agents included: phorbol myristate acetate (PMA), hydrogen peroxide (H2O2), N-formyl-methionyl-leucyl phenylalanine (FMLP), granulocyte-macrophage colony stimulating factor (GM-CSF), reduced glutathione (GSH), lipopolysaccharide (LPS) and interleukin 2 (IL-2). Apoptosis was assessed by traditional staining of the plates, by flow cytometric staining and DNA gel electrophoresis. It was found that without stimulation the susceptibility of PMN to apoptosis was slightly increased with aging. Under various stimulations, such as PMA. H2O2, apoptosis was almost 100%, while the treatment by FMLP, oxLDL and GSH did not change its extent in PMN obtained either from young or elderly subjects. Marked age-related changes were observed in the extent of apoptosis under stimulation with GM-CSF, IL-2 and LPS. These agents were able to significantly prevent apoptosis in PMN of young subjects, while only the GM-CSF was able to slightly modulate it in neutrophils of elderly subjects. From these results, we suggest that changes in apoptosis of PMN with aging could play a role in the increased incidence of certain immune system related pathologies of aging, such as cancer, infections and autoimmune disorders.

Adult↗